U.S. patent application number 16/609240 was filed with the patent office on 2020-06-18 for device and method for extracting nucleic acids from biological sample materials with solvent-free reagents.
This patent application is currently assigned to Siemens Healthcare Diagnostics Inc.. The applicant listed for this patent is Siemens Healthcare Diagnostics Inc.. Invention is credited to Walter Gumbrecht, Yiwei Huang, Peter Paulicka.
Application Number | 20200188910 16/609240 |
Document ID | / |
Family ID | 58701420 |
Filed Date | 2020-06-18 |
United States Patent
Application |
20200188910 |
Kind Code |
A1 |
Gumbrecht; Walter ; et
al. |
June 18, 2020 |
Device and method for extracting nucleic acids from biological
sample materials with solvent-free reagents
Abstract
The present invention relates to a device comprising a container
having an inner wall and at least one opening, containing a mixture
containing at least one reagent, and at least one additive, a
method of producing this device, and a method of extracting a
substance from a sample, enriching a substance in a sample, and/or
detecting a substance in a sample using the device.
Inventors: |
Gumbrecht; Walter;
(Herzogenaurach, DE) ; Huang; Yiwei; (Erlangen,
DE) ; Paulicka; Peter; (Rottenbach, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Siemens Healthcare Diagnostics Inc. |
Tarrytown |
NY |
US |
|
|
Assignee: |
Siemens Healthcare Diagnostics
Inc.
Tarrytown
NY
|
Family ID: |
58701420 |
Appl. No.: |
16/609240 |
Filed: |
April 24, 2018 |
PCT Filed: |
April 24, 2018 |
PCT NO: |
PCT/US2018/029048 |
371 Date: |
October 29, 2019 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 2300/0858 20130101;
B01L 3/5085 20130101; B01L 2200/12 20130101; B01L 3/5025 20130101;
G01N 33/54393 20130101; C12N 15/1013 20130101; B01L 2300/0851
20130101; B01J 20/26 20130101; B01L 2200/16 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12N 15/10 20060101 C12N015/10; G01N 33/543 20060101
G01N033/543; B01J 20/26 20060101 B01J020/26 |
Foreign Application Data
Date |
Code |
Application Number |
May 5, 2017 |
EP |
17169666.9 |
Claims
1. Device, comprising a container having an inner wall and at least
one opening, containing a mixture containing at least one reagent,
and at least one additive, wherein the at least one reagent is
fixed to at least part of the inner wall of the container with the
at least one additive, wherein the at least one reagent is at least
one of dispersed in the at least one additive, coated by the at
least one additive, and at least partially covered by the at least
one additive.
2. Device according to claim 1, wherein the at least one reagent
and the at least one additive are included in the container in the
form of a mixture that is essentially free of solvent.
3. Device according to claim 1, wherein no solvents are included in
the device.
4. Device according to claim 1, wherein the mixture comprises
amorphous and/or crystalline phases.
5. Device according to claim 1, wherein the container is a tube,
particularly a microtube, a well plate, or a bottle.
6. Device according to claim 1, wherein the at least one additive
is in solid form at room temperature.
7. Device according to claim 1, wherein the at least one additive
comprises a polymer, preferably a water-soluble polymer, further
preferably polyethyleneglycol, polypropyleneglycol,
polyvinylpyrrolidone and/or polyvinylalcohol, and/or copolymers
thereof.
8. Device according to claim 1, wherein the at least one reagent
comprises beads, particularly magnetic beads, and/or at least one
chaotropic agent, particularly a chaotropic salt, and/or at least
one detergent.
9. Device according to claim 1, wherein at least the inner wall of
the container comprises a polymeric material.
10. Method of extracting a substance from a sample, enriching a
substance in a sample and/or detecting a sub-stance in a sample,
comprising providing a device comprising a container having an
inner wall and at least one opening, containing a mixture
containing at least one reagent, and at least one additive, wherein
the at least one reagent is fixed to at least part of the inner
wall of the container with the at least one additive, wherein the
at least one reagent is at least one of dispersed in the at least
one additive, coated by the at least one additive, and at least
partially covered by the at least one additive; introducing the
sample into the container; and mixing the sample with the mixture
containing the at least one reagent, and the at least one
additive.
11. Method of claim 10, wherein the mixing is carried out using
mechanical agitation and/or a temperature increase.
12. Method of claim 10, wherein the sample is an aqueous
sample.
13. Method of claim 10, wherein the substance is a nucleic
acid.
14. Method of producing a device, comprising: providing a container
having an inner wall and at least one opening; preparing a mixture
containing at least one reagent and at least one additive; and
introducing the mixture in the container, wherein the at least one
reagent is fixed to at least part of the inner wall of the
container with the at least one additive, wherein the at least one
reagent is at least one of dispersed in the at least one additive,
coated by the at least one additive, and at least partially covered
by the at least one additive.
15. Method of claim 14, wherein the mixture is introduced in the
form of a melt and the melt is solidified, or wherein the mixture
is introduced in the form of a paste, or wherein the mixture is
introduced in the form of a pow-der mixture and subsequently heated
in the container, or wherein the at least one reagent is introduced
into the container and subsequently coated with the at least one
additive.
Description
[0001] The present invention relates to a device comprising a
container having an inner wall and at least one opening, containing
a mixture containing at least one reagent, and at least one
additive, a method of producing this device, and a method of
extracting a substance from a sample, enriching a substance in a
sample, and/or detecting a substance in a sample using the
device.
[0002] In molecular diagnostics sample preparation is of particular
importance since--in contrast to clinical-chemical (CC) or
immunoassays (IA)--the sample material, e.g. blood plasma, cannot
be examined directly, but a material to be examined, e.g. a nucleic
acid, has to be first extracted, e.g. from human cells, bacteria or
viruses, and cleaned.
[0003] A number of established techniques exist for this purpose,
which are based on the usage of e.g. detergents (membrane lysis),
chaotropic salts (protein denaturation), proteases (enzymatic
digestion), and magnetic beads, e.g. silica beads (bonding of
nucleic acids). In all cases the reagents are present in liquid or
dissolved/suspended form (mostly aqueous). This involves extensive
pipetting steps in these methods, respectively workflows, for
nucleic acid extraction being necessary, either manually or
automated. This leads to a high usage of pipette tips (cost, waste)
and an enormous amount of time spent. Also logistical requirements,
like cool storage, expiry dates, and treatment of opened packages
or containers, have to be considered.
[0004] A further problem is based on requirements regarding the
usage of reagents. For example, chaotropic agents, e.g. salts like
guanidinium thiocyanate, have to be used in very high final
concentrations in presence of a sample liquid, but the solubility
thereof in the reagent solution is limited. As a consequence a high
reagent volume (V.sub.r) is necessary for treating a certain sample
volume (V.sub.p), which can surpass it by a manifold
(V.sub.r>V.sub.p). A consequence thereof is a reduced sample
volume and therefore a reduced yield.
[0005] A partial simplification of the work flow and logistics can
be achieved for some producers by providing reagents necessary for
processing a single sample in liquid form in individual
compartments of a plastic cassette, e.g. reagent cassettes with
liquid reagents for nucleic acid extraction, e.g. by Roche MagNA
Pure and Becton Dickinson BD MAX.TM.. For removing the reagent,
seal membranes thereof can be e.g. pierced by a pipette tip.
However, this approach does not solve the problems of logistics and
limiting of sample volume.
[0006] Therefore there is a need for improved methods and devices
for extracting a substance from a sample and/or enriching in a
sample and/or detecting a substance in a sample, wherein
particularly the amount of pipetting and/or solvents is
reduced.
[0007] The inventors found a device for extracting a substance from
a sample and/or enriching in a sample and/or detecting a substance
in a sample in which the use of pipetting steps and solvent use can
be minimized or--in case of solvent use--even avoided by fixing the
reagents in a container inside a matrix of an additive.
[0008] With the present device the need for a solvent like water in
the above procedures can be reduced or even eliminated, e.g. when
using reagents, particularly when the sample itself contains a
liquid like water. For example, in blood sample testing blood
plasma consists of about 90 wt. % of water.
[0009] Furthermore, also the storage stability of the reagents can
be improved due to avoiding solvents. In addition, no measures are
necessary to avoid evaporation of solvents, making the procedures
easier and more reliable. Also for analytical purposes this is of
particular importance. Apart from that, also logistics can be
simplified as a sample can be used as is and pipetting steps can be
reduced. Particularly, pipetting of reagent solutions can be
reduced or even totally avoided, reducing the amount of time, tips,
and waste in the procedures. Apart from that, bigger sample amounts
are possible to use with constant reagent concentration.
[0010] In a first aspect the present invention relates to a device
comprising a container having an inner wall and at least one
opening, containing a mixture containing at least one reagent and
at least one additive, wherein the at least one reagent is fixed to
at least part of the inner wall of the container with the at least
one additive, wherein the at least one reagent is dispersed in the
at least one additive, and/or the at least one reagent is coated by
the at least one additive, and/or the at least one reagent is at
least partially covered by the at least one additive.
[0011] Furthermore discloses is a method of extracting a substance
from a sample, enriching a substance in a sample and/or detecting a
substance in a sample, comprising [0012] providing the present
device; [0013] introducing the sample into the container; and
[0014] mixing the sample with the mixture containing the at least
one reagent, and the at least one additive.
[0015] In addition, a further aspect relates to a method of
producing the present device, comprising: [0016] providing a
container having an inner wall and at least one opening; [0017]
preparing a mixture containing at least one reagent and at least
one additive; and [0018] introducing the mixture in the container,
wherein the at least one reagent is fixed to at least part of the
inner wall of the container with the at least one additive, wherein
the at least one reagent is dispersed in the at least one additive,
and/or the at least one reagent is coated by the at least one
additive, and/or the at least one reagent is at least partially
covered by the at least one additive.
[0019] Further aspects and embodiments of the invention are
disclosed in the dependent claims and can be taken from the
following description, figures and examples, without being limited
thereto.
FIGURES
[0020] The enclosed drawings should illustrate embodiments of the
present invention and convey a further understanding thereof. In
connection with the description they serve as explanation of
concepts and principles of the invention. Other embodiments and
many of the stated advantages can be derived in relation to the
drawings. The elements of the drawings are not necessarily to scale
towards each other. Identical, functionally equivalent and acting
equal features and components are denoted in the figures of the
drawings with the same reference numbers, unless noted
otherwise.
[0021] FIG. 1 shows a schematic drawing of an embodiment of the
present invention.
[0022] FIG. 2 shows schematically the requirements of a method of
the prior art.
[0023] FIG. 3 shows an Example of the present device.
DETAILED DESCRIPTION OF THE INVENTION
Definitions
[0024] Unless defined otherwise, technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs.
[0025] In the context of the present invention a "sample" is a
sample to be analyzed for a substance and/or which comprises a
substance to be extracted and/or enriched, e.g. at least an analyte
and/or a nucleic acid. According to certain embodiments the
substance in the present method of extracting a substance from a
sample, enriching a substance in a sample and/or detecting a
substance in a sample is a nucleic acid and/or an analyte, e.g. a
nucleic acid, particularly when the sample is extracted and/or
enriched, or an analyte when the sample is analyzed.
[0026] The sample is not particularly restricted and can be of
natural or synthetic origin and can e.g. comprise samples from
simple or complex matrices, e.g. from a subject, e.g. a vertebrate,
e.g. a human or animal, or from a natural source, e.g. an extract
from a soil, air, or a natural water/water body. The sample can
also be water-free or water-poor.
[0027] According to certain embodiments the sample is an aqueous
sample, i.e. a sample containing water. For example, the sample can
be a body fluid of a subject. Body fluids are thereby liquids
originating from inside the bodies of subjects, particularly living
subjects. They include fluids that can be excreted or secreted from
the body and/or that circulate in the body, and body water. They
can be in the state of a liquid, emulsion or suspension. Examples
of body fluids within the invention are blood, urine, saliva,
sputum, plasma, serum, etc. According to certain embodiments, the
sample is a patient sample (clinical isolate). Exemplified samples
are serum, plasma, and/or whole blood of a patient.
[0028] According to certain embodiments, the subject in the present
method of extracting a substance from a sample, enriching a
substance in a sample and/or detecting a substance in a sample can
be a vertebrate, preferably a human or animal, more preferably a
mammal and most preferred a human, respectively human patient.
[0029] A vertebrate within the present invention refers to animals
having a vertebrate, which includes mammals--including humans,
birds, reptiles, amphibians and fishes. The present invention thus
is not only suitable for humans and the human medical field, but
also for veterinary medicine.
[0030] The term "nucleic acid" refers to a polynucleotide molecule
having a defined sequence. It comprises DNA molecules, RNA
molecules, nucleotide analog molecules and combinations and
derivatives thereof, such as DNA molecules or RNA molecules with
incorporated nucleotide analogs or cDNA. It also comprises cell
free (cf) DNA and RNA. The term "nucleic acid sequence" relates to
the sequence of nucleotides in the nucleic acid molecule.
[0031] Before the invention is described in exemplary detail, it is
to be understood that this invention is not limited to the
particular component parts of the process steps of the methods
described herein as such methods may vary. It is also to be
understood that the terminology used herein is for purposes of
describing particular embodiments only, and is not intended to be
limiting. It must be noted that, as used in the specification and
the appended claims, the singular forms "a," "an" and "the" include
singular and/or plural referents unless the context clearly
dictates otherwise. For example, the term "a" as used herein can be
understood as one single entity or in the meaning of "one or more"
entities. It is also to be understood that plural forms include
singular and/or plural referents unless the context clearly
dictates otherwise. It is moreover to be understood that, in case
parameter ranges are given which are delimited by numeric values,
the ranges are deemed to include these limitation values. The term
"essentially" when used herein means to an extent of greater than
70%, preferably greater than 80%, even more preferred greater than
90% and most preferred greater than 95%.
[0032] A first aspect of the present invention relates to a device
comprising a container having an inner wall and at least one
opening, containing a mixture containing [0033] at least one
reagent, and [0034] at least one additive,
[0035] wherein the at least one reagent is fixed to at least part
of the inner wall of the container with the at least one additive,
wherein the at least one reagent is dispersed in the at least one
additive, and/or the at least one reagent is coated by the at least
one additive, and/or the at least one reagent is at least partially
covered by the at least one additive.
[0036] With regard to the present invention, the inner wall of the
container is a wall that can come into contact with the mixture
containing at least one reagent and at least one additive, as well
as with a sample that is introduced into the container. It can
comprise e.g. the bottom in the inside of the container as well as
inner side walls of the container. This means that the mixture
containing at least one reagent and at least one additive can be
fixed at the bottom and/or at the side walls in the inside of the
container.
[0037] The container is not particularly restricted and can be in
any form as long as it provides an inner wall and at least one
opening, preferably one opening. It can be out of any suitable
material and can take up any volume suitable for introducing a
sample therein. It can be e.g. a tube, a well plate, a bottle, a
beaker, a flask, etc., and can be in any shape. Preferably, the
container has a volume that can be introduced of less than 500 mL,
further preferably less than 200 mL, even further preferably less
than 100 mL, e.g. 50, 20, 10, 5 or 2 mL or less.
[0038] According to certain embodiments the container is a tube,
e.g. a test tube or a microtube, particularly a microtube, a
cuvette, a well in a well plate, e.g. a microtiter plate, e.g. a
96-well titer plate; or a bottle. The device thus can be, according
to certain embodiments, a well plate, e.g. a microtiter plate, e.g.
a 96-well titer plate. According to certain embodiments, the
container is not a dish, e.g. a Petri dish. Particularly tubes
and/or cuvettes are advantageous as containers for use in e.g.
high-throughput analyzation, extraction and/or enrichment, as a
multitude of tubes and/or cuvettes, e.g. without a cap, can easily
be filled, e.g. as bulk, respectively in loose form, into a
high-throughput machine, e.g. a high-throughput analyzer, where
they can be sorted and put upright before filling the device with a
sample to be extracted, enriched and/or analyzed.
[0039] The container can be made of any material, e.g. ceramic,
glass, metal, plastics, etc., as long as the material is suitable
to take up the mixture containing at least one reagent and at least
one additive, and preferably also suitable to have a sample to be
analyzed, extracted and/or enriched introduced therein. Also,
different parts of the container can be made of different material,
e.g. an inner and an outer wall, or be made of the same material.
It is also not excluded that the container or parts thereof are
coated by a material before introducing the mixture containing at
least one reagent and at least one additive. However, the
container, or parts thereof, can also be uncoated. If a coating is
provided the material for the coating is not particularly limited,
and can e.g. comprise a substance for binding and/or fixing an
analyte in a sample to be analyzed and/or enriched and/or
extracted. For example, a coating can be provided at least on the
inner wall that enhances the fixing of the at least one reagent to
the inner wall of the container with the at least one additive,
e.g. a material that can physically and/or chemically interact with
the at least one additive. According to certain embodiments, the
material of the container and/or optionally a coating is inert
towards the mixture containing the at least one reagent and the at
least one additive, and/or optionally towards a sample to be
analyzed and/or enriched and/or extracted.
[0040] According to certain embodiments at least the inner wall of
the container comprises a polymeric material. It is also possible
that the further container comprises a polymeric material or that
the whole container consists of a polymeric material, e.g. a
thermoplastic material like polyethylene, polypropylene,
polystyrene, polyvinylchloride, or mixtures or copolymers thereof.
With the polymeric material an improved fixation of the at least
one reagent to at least part of the inner wall of the container
with the at least one additive is possible, e.g. by adhesion and/or
van der Waals force. However, the container can also comprise glass
or consist of glass.
[0041] The fixing of the at least one reagent to at least part of
the inner wall of the container with the at least one additive is
not particularly restricted as long as it can be avoided that the
at least one reagent moves in the container and/or is dropped from
the container when the container is moved, e.g. turned upside down,
particularly when the container is centrifuged, e.g. upside down
(with the at least one opening facing the ground) for 5 mins at a
centrifugal force of 100 g, e.g. with a Hettich Universal 320 R
centrifuge when the container is a 2 mL Eppendorf tube that is put
upside down in a 15 mL Falcon tube. According to certain
embodiments, the mixture is fixed to at least part of the inner
wall of the container in such a way that no components thereof are
leaving the container, particularly a lump formed by the mixture,
when the container is moved, e.g. turned upside down, particularly
when the container is centrifuged, e.g. upside down (with the at
least one opening facing the ground) for 5 mins at a centrifugal
force of 100 g, e.g. with a Hettich Universal 320 R centrifuge when
the container is a 2 mL Eppendorf tube that is put upside down in a
15 mL Falcon tube.
[0042] By fixing the at least one reagent to the at least part of
the inner wall of the container with the at least one additive the
at least one reagent is not diluted by a solvent, which is
especially of advantage for reagents which have to be used in a
high concentration, e.g. 1-5 molar in water, e.g. 3-4 molar in
water, in a method of extracting a substance from a sample,
enriching a substance in a sample and/or detecting a substance in a
sample, e.g. chaotropic salts in a method of extracting nucleic
acids from a sample, e.g. blood sample.
[0043] The mixture containing the at least one reagent, and the at
least one additive is not particularly restricted as long as both
the at least one reagent and the at least one additive are present
in a combined form in the container. It is not excluded that they
form separate phases, as long as both are in contact with each
other and as long as the at least one reagent is fixed to at least
part of the inner wall of the container with the at least one
additive.
[0044] In the invention, the at least one reagent is dispersed in
the at least one additive, and/or the at least one reagent is
coated by the at least one additive, and/or the at least one
reagent is at least partially covered by the at least one additive.
This means that the at least one reagent forms a mixture with the
at least one additive such that the at least one reagent is
enclosed in, embedded in, respectively coated by the at least one
additive, at least in part. The reagent therein preferably does not
chemically react with the at least one additive when forming the
mixture and/or when put into the container and/or during transport,
storage, etc. Alternatively, it is also possible that the at least
one reagent is returned to its original state when applying a
sample to the mixture containing the at least one reagent, and the
at least one additive. However, preferably the at least one
additive does not change the at least one reagent when the mixture
is formed and/or when the at least one reagent is dispersed in the
at least one additive, and/or the at least one reagent is coated by
the at least one additive, and/or the at least one reagent is at
least partially covered by the at least one additive.
[0045] The formation of a dispersion is not particularly restricted
as well as the state of dispersion of the at least one reagent in
the at least one additive, and it is not excluded that some part of
the reagent is in contact with the inner wall of the container
and/or that a bigger volume of one or more reagents forms. It is
also possible that the at least one reagent is dispersed in the at
least one additive. Alternatively or in addition, also the coating
of the at least one reagent by the at least one additive is not
restricted, and it is possible that the whole reagent is coated by
the at least one additive, also e.g. in such a way that the at
least one reagent is coated as a whole by the at least one additive
or is coated in several parts by the at least one additive, wherein
the at least one reagent again also can be--at least in part--in
contact with the inner wall of the container, e.g. also the bottom.
Alternatively or in addition, also the covering of the at least one
reagent by the at least one additive is not particularly
restricted, and it is also possible that at least some part of the
at least one reagent is only partially covered by the at least one
additive, e.g. in the direction of the at least one opening of the
container, e.g. an upper surface of the mixture, as long as the at
least one reagent is sufficiently retained by the at least one
additive, e.g. if it is covered to a large extent, e.g. in the case
of reagent particles.
[0046] A schematic drawing of a container 1 having an inner wall 2
containing a mixture 3 of a reagent 4 and an additive 5 is shown in
FIG. 1. In this figure the reagent 4 is covered in whole by the
additive 5 and thus forms a dispersion of the reagent 4 in the
additive 5. FIG. 1 thus shows a schematic view of a device
according to the present invention. While the container 1 is shown
in FIG. 1 in the form of a tube with a round bottom, it is not
restricted to such a shape, though.
[0047] According to certain embodiments the at least one reagent
and the at least one additive are included in the container in the
form of a mixture that is essentially free of solvent. This does
not exclude a case wherein at least one reagent is provided in
liquid form, wherein e.g. liquid droplet are dispersed in the at
least one additive. It is also not excluded that a solvent
functions as at least one reagent, but such a case is not
preferred, and the mixture of the at least one reagent and the at
least one additive is preferably free of solvent. A solvent is this
regard is a material that can dissolve another substance and can be
an inorganic or organic, polar or non-polar solvent, e.g. water
and/or an organic solvent like an alkane, an alcohol, an ester, an
ether, a ketone, an amine, etc., and is not particularly
restricted. A particular solvent is a polar solvent, e.g.
water.
[0048] According to certain embodiments no solvents are included in
the device. This means that the mixture as well as the container
and any other optional parts of the device do not include a
solvent. For example, the present device can be suitably packed or
at least the at least one opening suitably closed, e.g. sealed, to
avoid any ingress of a solvent, e.g. water.
[0049] According to certain embodiments the mixture comprises
amorphous and/or crystalline phases. These can then be at least
attributed to the at least one additive, which can form a solid,
e.g. at a suitable temperature, e.g. room temperature, e.g.
20-25.degree. C., e.g. 20, 21, 22, 23, 24 or 25.degree. C., in
which the at least one reagent or more than one reagents are fixed.
In this instance the reagents themselves do not have to be solid
and can be e.g. also liquid at that temperature, e.g. room
temperature, e.g. 20-25.degree. C., e.g. 20, 21, 22, 23, 24 or
25.degree. C., e.g. in the case of detergents.
[0050] According to certain embodiments the at least one additive
is in solid and/or liquid form of sufficient dynamic viscosity p of
e.g. more than 1*10.sup.4 mPas, e.g. more than 1*10.sup.5 mPas,
1*10.sup.6 mPas, 1*10.sup.7 mPas, or 1*10.sup.8 mPas at room
temperature when measured with a plate rheometer, e.g. is in solid
form, at room temperature, e.g. 20-25.degree. C., e.g. 20, 21, 22,
23, 24 and/or 25.degree. C. However, the at least one additive can
still be ductile at room temperature, i.e. can be deformed, e.g.
like a thermoplastic, or like a wax at 20.degree. C. that is
kneadable. According to certain embodiments, the at least one
additive is ductile at a temperature between -30 and 60.degree. C.,
e.g. between -20 and 50.degree. C., e.g. between -10 and 45.degree.
C., e.g. between 0 and 40.degree. C., e.g. at least of
20-25.degree. C., e.g. 20, 21, 22, 23, 24 and/or 25.degree. C. When
the at least one additive is in liquid form of sufficient
viscosity, the at least one opening of the container can also be
covered, e.g. by a foil, e.g. a protection foil. According to
certain embodiments, the device can be stored for a sufficient time
at a temperature between -30 and 60.degree. C., e.g. between -20
and 50.degree. C., e.g. between -10 and 45.degree. C., e.g. between
0 and 40.degree. C., e.g. at least of 20-25.degree. C., e.g. 20,
21, 22, 23, 24 and/or 25.degree. C. for a sufficient time, e.g. at
least one week, at least one month or at least one year, e.g. also
upside-down with the at least one opening facing towards the ground
without the mixture or a part thereof leaving the container,
preferably without the mixture changing the shape thereof in the
container.
[0051] According to certain embodiments the at least one additive
comprises a polymer, preferably a water-soluble polymer, further
preferably polyethylene glycol (PEG), polypropylene glycol (PPG),
polyvinylpyrrolidone and/or polyvinyl alcohol, and/or copolymers
thereof. According to certain embodiments, the at least one
additive contains polyethylene glycol or is polyethylene glycol.
According to certain embodiments, the molecular weight of the
polymer is set in a range so that it is ductile at a temperature
between -30 and 60.degree. C., e.g. between -20 and 50.degree. C.,
e.g. between -10 and 45.degree. C., e.g. between 0 and 40.degree.
C., e.g. at least of 20-25.degree. C., e.g. 20, 21, 22, 23, 24
and/or 25.degree. C., e.g. to a weight average molecular weight
between 500 and 10000, e.g. between 1000 and 8000, e.g. to about
6000, for polyethylene glycol. According to certain embodiments,
the mixture containing the at least one reagent and the at least
one additive, is ductile at a temperature between -30 and
60.degree. C., e.g. between -20 and 50.degree. C., e.g. between -10
and 45.degree. C., e.g. between 0 and 40.degree. C., e.g. at least
of 20-25.degree. C., e.g. 20, 21, 22, 23, 24 and/or 25.degree. C.,
to a degree that it does not separate from the at least part of the
inner wall of the container during storage for a certain time, e.g.
at least one week, at least one month or at least one year, e.g.
also due to shrinkage.
[0052] With these additives, an improved binding of a substance to
be enriched and/or extracted can e.g. be achieved, e.g. in the case
of nucleic acids from a sample, e.g. by improving the bonding
properties of nucleic acids to silica beads.
[0053] By selecting an additive with suitable properties (e.g. PEG
with suitable molecular weight (weight average molecular weight)
and therefore suitable melting point of e.g. 30 to 100.degree. C.,
e.g. of 40 to 90.degree. C., e.g. of 50 to 75.degree. C., e.g. of
about 60.degree. C.) the mixture of reagents and additive can be
introduced, e.g. pressed, as a paste and/or at an increased
temperature, e.g. 30-200.degree. C., e.g. 35-180.degree. C., e.g.
37-155.degree. C., 38 to 150.degree. C., e.g. 40 to 120.degree. C.,
e.g. 50 to 95.degree. C., into the container.
[0054] According to certain embodiments, the at least one additive,
e.g. polyethylene glycol, has a solubility in water of at least 33
g/L at a temperature of 15.degree. C. to 25.degree. C., e.g. of 20
25.degree. C., e.g. 20, 21, 22, 23, 24 and/or 25.degree. C., at
normal pressure, i.e. 101325 Pa, preferably of at least 100 g/L,
further preferably of at least 1000 g/L.
[0055] According to certain embodiments, the at least one additive
can form a "wax-like" coating around the at least one reagent, e.g.
particles, e.g. (crystal) particles of a chaotropic salt, and/or
beads, e.g. magnetic silica-beads.
[0056] According to certain embodiments the at least one additive
can form a coating around and/or a suspension with and/or a
solution with a detergent and/or emulsifier, e.g. a liquid
detergent and/or emulsifier like octoxinol 9 (Triton X-100) and/or
polysorbates like Tween.RTM. emulsifiers, and/or can absorb the
detergent and/or emulsifier, e.g. a liquid detergent and/or
emulsifier like octoxinol 9 (Triton X-100) and/or polysorbates like
a Tween.RTM. emulsifier, e.g. Tween.RTM. 20, e.g. by solving
thereof, preferably while keeping the "wax-like" properties.
[0057] According to certain embodiments, the at least one additive
shows good adhesion to the inner wall of the container (e.g.
polyethylene, polypropylene, polystyrene, polyvinylchloride, or
mixtures or copolymers thereof). According to certain embodiments,
the at least one additive is elastic and/or not brittle and/or
shows no danger of cracking at a temperature between -30 and
60.degree. C., e.g. between -20 and 50.degree. C., e.g. between -10
and 45.degree. C., e.g. between 0 and 40.degree. C., e.g. at least
at room temperature, e.g. 20, 21, 22, 23, 24 and/or 25.degree. C.,
and normal pressure.
[0058] In the present invention the at least one reagent is not
particularly limited and can comprise one reagent or a mixture of
two or more, e.g. three of more or four or more, reagents. If more
than one reagent is comprised it is possible that different
reagents can be contained in different layers of mixtures of
additive and reagent, e.g. by introducing the different mixtures of
at least one additive and at least one reagent one after the other
into the container, e.g. layering them, so that different reagents
can come into contact with the sample at different times upon
introduction of the sample. In such an instance it is also possible
that different additives are used in the different layers that are
dissolved by a liquid of the sample at different times.
[0059] The type of reagent is not particularly limited and can
comprise any reagent that can interact with a substance in a sample
to be analyzed, enriched and/or extracted and/or support the
interaction with the sample. "Interact" means the capability to
chemically react, chemically cause a reaction, biologically bind
to/with a partner, while the reagent is typically consumed in the
course of the reaction (as opposed to a solvent or a catalyst). It
can comprise antibodies, markers, enzymes, beads, detergents,
emulsifiers, oligomers, nucleic acids and/or derivatives thereof,
chaotropic agents, an internal control, etc. The reagents can be in
the form of particles, liquids, beads, emulsions, powder, or any
other suitable form for interacting with the sample upon use of the
device and dissolution of the at least one additive.
[0060] According to certain embodiments the at least one reagent
comprises beads, particularly magnetic beads, e.g. magnetic silica
beads, and/or at least one chaotropic agent like a chaotropic salt,
urea, thiourea, etc., particularly a chaotropic salt like sodium
dodecyl sulfate, lithium perchlorate, lithium acetate, magnesium
chloride or a guanidinium salt, e.g. guanidinium chloride and/or
guanidinium thiocyanate, and/or at least one detergent. According
to certain embodiments, beads, e.g. magnetic silica beads, at least
one chaotropic agent like a chaotropic salt, e.g. a guanidinium
salt, and at least one detergent, e.g. a polysorbate, are used as
reagent. With this combination an extraction of nucleic acids from
a sample, e.g. blood sample, is possible. According to certain
embodiments, the at least one reagent contains beads, e.g. magnetic
silica beads. According to certain embodiments, the at least one
reagent contains a chaotropic agent, e.g. a chaotropic salt.
According to certain embodiments, the at least one reagent contains
a detergent and/or an emulsifier.
[0061] According to certain embodiments, the at least one reagent
and/or the at least one additive do not have a notable vapor
pressure, e.g. a vapor pressure below 2 kPa, e.g. below 1.8 kPa,
e.g. below 1.7 kPa, e.g. below 1.6 kPa, e.g. below 1.5 kPa, e.g.
below 1.4 kPa, e.g. also below 1 kPa, e.g. as measured by a Knudsen
cell.
[0062] According to certain embodiments, the mixture of the at
least one reagent and the at least one additive can form a compact
phase with a solid outer surface. According to certain embodiments,
this compact phase does not notably dissolve, e.g. with less than
10 g/L sample, e.g. less than 1 g/L sample, e.g. less than 0.1 g/L
sample, for several minutes, e.g. 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10
minutes, at room temperature, e.g. of 20-25.degree. C., e.g. 20,
21, 22, 23, 24 and/or 25.degree. C., upon addition of an aqueous
sample without agitation of the mixture and/or sample. According to
certain embodiments, the compact phase or at least the at least one
additive dissolves upon addition of an aqueous sample and agitation
and/or a temperature increase, e.g. to a temperature of 30 to
100.degree. C., e.g. of 40 to 90.degree. C., e.g. of 50 to
75.degree. C., e.g. more than 60.degree. C.
[0063] According to certain embodiments, the present device is a
device for extracting nucleic acids from biological samples,
consisting of: [0064] a container for receiving the sample, i.e. a
sample material, and [0065] reagents that are fixed with an
additive to an inner wall of the container.
[0066] A further aspect of the present invention relates to a
method of extracting a substance from a sample, enriching a
substance in a sample and/or detecting a substance in a sample,
comprising [0067] providing the device of the present invention, as
disclosed above; [0068] introducing the sample into the container,
e.g. filling the container with the sample, e.g. an aqueous sample
like a blood sample or a sample derived therefrom; and [0069]
mixing the sample with the mixture containing the at least one
reagent, and the at least one additive.
[0070] According to certain embodiments the mixing is carried out
using mechanical agitation and/or a temperature increase, e.g. to a
temperature of 30 to 100.degree. C., e.g. of 40 to 90.degree. C.,
e.g. of 50 to 75.degree. C., e.g. of about 60.degree. C.
[0071] For introducing the sample to the container, an addition of
a solvent like water to a sample is also possible prior to the
introduction if the sample is a water-free or water-poor
sample.
[0072] An exemplary embodiment of the present method of extracting
a substance from a sample, enriching a substance in a sample and/or
detecting a substance in a sample, here extracting nucleic acids
from a blood sample, is shown with regard to FIG. 3, whereas FIG. 2
shows requirements needed with regard to a method thereof of the
state of the art.
[0073] In FIGS. 2 and 3, the exemplary container is a 96-well titer
plate, here a deep well plate 16, wherein the sample is introduced
into the wells thereof, e.g. after introducing the mixture
containing at least one reagent and at least one additive in the
case of the present method.
[0074] A lysis buffer 11, beads 12 and an internal control 13 are
exemplified as necessary agents in the embodiment shown in FIG. 2.
These have to be decanted separately from screw-cap bottles to
pipetting containers and then pipetted into the 96 wells using tips
14, e.g. 140 tips on average for a suitable extraction, taking
about 30 minutes in pipetting step 15.
[0075] In contrast, in the present method the mixture 3 containing
the at least one reagent, e.g. the lysis buffer, beads and the
internal control in case of the method shown in FIG. 2, and the at
least one additive is introduced into the wells of the 96-well
titer plate, wherein each well can be seen as a container 1, but
also the whole plate can be seen as a container having 96 openings,
as depicted in FIG. 3. Thus, the amount of liquid in the system can
be minimized as well as the pipetting amount, with only one step of
pipetting for introducing the sample in each respective well
necessary. This way also the number of pipetting tips can be
reduced.
[0076] Even filling of the 96-well plate with sample material takes
several minutes e.g. with a 8-channel pipetting robot. This leads
also to different starting times for the separate wells during
extracting and/or enriching and/or analysis, which has to be
accounted for in a quantitative method.
[0077] However, as the mixture of the at least one reagent and the
at least one additive can form a compact phase with a solid outer
surface in the present device, all 96 samples in the 96-well titer
plate can be incubated and/or reacted at about the same time with
the reagents, e.g. by starting a simultaneous agitation and/or
temperature increase after introducing sample in all wells.
[0078] Furthermore disclosed in another aspect of the present
invention is a method of producing the present device, as discloses
above, comprising: [0079] providing a container having an inner
wall and at least one opening; [0080] preparing a mixture
containing at least one reagent and at least one additive; and
[0081] introducing the mixture in the container,
[0082] wherein the at least one reagent is fixed to at least part
of the inner wall of the container with the at least one additive,
wherein the at least one reagent is dispersed in the at least one
additive, and/or the at least one reagent is coated by the at least
one additive, and/or the at least one reagent is at least partially
covered by the at least one additive.
[0083] With this method the device of the present invention can be
prepared. Thus, all features discussed with regard to the present
device apply also to this method.
[0084] In this method, the preparation of the mixture containing at
least one reagent and at least one additive is not particularly
restricted, and the mixture can be prepared in any way possible,
e.g. as a powder mixture, a suspension which can be "solidified" in
the container, a mix, etc.
[0085] According to certain embodiments the mixture is introduced
in the form of a melt and the melt is solidified. The forming of a
melt is not particularly restricted, and a melt can be formed by
e.g. heating the mixture containing the at least one reagent and
the at least one additive to a suitable temperature, e.g. depending
on the additive, wherein it is not excluded that some parts of the
mixture are not molten. A suitable temperature can be e.g.
30-200.degree. C., e.g. 50-180.degree. C., e.g. 70-155.degree. C.,
e.g. 75-130.degree. C., e.g. 80 to 120.degree. C.
[0086] According to certain embodiments the mixture is introduced
in the form of a paste. Also the formation of the paste is not
particularly restricted. For example, the at least one reagent can
be introduced into a sufficiently ductile additive, e.g. by
kneading, shaking, etc. For example, by selecting at least one
additive with suitable properties (e.g. PEG with suitable molecular
weight (weight average molecular weight) and therefore suitable
melting point, as described above), the mixture of the at least one
reagent and the at least one additive can be pressed as paste, e.g.
at increased temperature of 30 to 80.degree. C., e.g. of 40 to
75.degree. C., e.g. of 50 to 70.degree. C., e.g. of about
60.degree. C., for example, into the container.
[0087] According to certain embodiments the mixture is introduced
in the form of a powder mixture and subsequently heated, e.g.
molten, in the container, e.g. by heating for a suitable time and
at a suitable temperature, which can depend e.g. on the at least
one additive. Subsequently the melt can be solidified, e.g. form a
compact phase which is solid. A suitable temperature can be e.g.
30-200.degree. C., e.g. 50-180.degree. C., e.g. 70-155.degree. C.,
e.g. 75-130.degree. C., e.g. 80 to 120.degree. C., and a suitable
time between 30 seconds and 60 minutes, e.g. between 1 and 50
minutes, e.g. between 3 and 45 minutes, e.g. between 5 and 40
minutes, e.g. between 7 and 30 minutes.
[0088] According to certain embodiments the at least one reagent is
introduced into the container and subsequently coated with the at
least one additive. In such instance reagents can be introduced
separately or as a mixture, e.g. a powder mixture, into the
container if more than one reagent is introduced. The coating with
the at least one additive is also not restricted, and the at least
one additive can be introduced as powder and then melted in the
container, e.g. as described above with regard to the introduction
in the form of a powder mixture, or introduced as a melt, e.g. as
described above with regard to the introduction in the form of a
melt. It is not excluded that the mixture is then further heated
until a compact phase is generated.
[0089] The above embodiments can be combined arbitrarily, if
appropriate. Further possible embodiments and implementations of
the invention comprise also combinations of features not explicitly
mentioned in the foregoing or in the following with regard to the
Examples of the invention. Particularly, a person skilled in the
art will also add individual aspects as improvements or additions
to the respective basic form of the invention.
EXAMPLES
[0090] The present invention will now be described in detail with
reference to several examples thereof. However, these examples are
illustrative and do not limit the scope of the invention.
Example 1
[0091] A magnetic silica-bead-suspension of Siemens VERSANT sample
preparation 1.0 reagents kit (beads), Guanidine thiocyanate,
BioUltra, SIGMA (GuSCN) (rubbed to powder in a porcelain mortar),
and Tween.RTM. 20, molecular biology grade, VWR (Tween) were added
as reagents into a 2 ml Eppendorf tube (Eppi) together with
poly(ethylene glycol) 6000, BioUltra, Fluka (PEG) (rubbed to powder
in a porcelain mortar) as an additive, in the following manner.
[0092] 25 .mu.L beads are pipetted to the Eppendorf tube,
magnetized and the solvent is pipetted off. 430 mg GuSCN are added,
65 mg PEG are added, and 50 mg Tween are added. The mixture is
heated for 20 mins at 90.degree. C. Then it is centrifuged hot at
12000 rpm in the tube in a miniSpin centrifuge for 1 min.
[0093] The surface of the mixture in the Eppendorf tube is slightly
rough, GuSCN is coated by the PEG, and amorphous and crystalline
phases can be observed.
[0094] After cooling the tube with the mixture, it is centrifuged
"upside down" with a Hettich Universal 320 R centrifuge (the 2 mL
Eppendorf tube with the mixture is put upside down in 1 15 mL
Falcon tube) for 5 mins at a centrifugal force of 100 g. With such
centrifugation no change is observed for the mixture in the
Eppendorf tube. After 5 mins in the same centrifuge at 200 g the
mixture is detached from the tube.
[0095] This shows that the mixture can be suitably fixed to the
Eppendorf tube for storage.
Example 2
[0096] A tube as prepared in Example 1 was used for testing
dissolution of the mixture in a sample. As sample, 500 .mu.L blood
plasma were pipetted on the surface of the mixture in the Eppendorf
tube. The sample was then left for 5 minutes, during which time no
visible change was observed for the mixture.
[0097] Thereafter, the tube with the sample liquid was put in a
heater-shaker at a temperature of 60.degree. C. and at 1000 rpm.
Within 5 minutes the mixture completely dissolved in the blood
plasma.
[0098] Thereafter, the nucleic acids in the blood plasma were
enriched in a usual manner with the reagents contained in the
mixture.
* * * * *